The publications referred to in what follows and identified by footnotes are incorporated by reference herein for their teachings as to capillary electrophoretic techniques.
Capillary electrophoresis (CE) has been established as an important separation method in bioanalytical chemistry. Separation and detection of very small amounts of biological samples, about pL-nL volumes, can be achieved with CE. This is generally not possible with more conventional separatory methods, even high performance liquid chromatography (HPLC). There are several CE separation methods in use for different kinds of samples. They include capillary zone electrophoresis, moving boundary capillary electrophoresis, capillary isotachophoresis and capillary isoelectric focusing.
Isoelectric focusing (IEF) is a special electrophoretic technique for separating amphoteric substances such as peptides and proteins in an electric field, across which there is both a voltage and a pH gradient, acidic in the region of the anode and alkaline near the cathode. Each substance in the mixture will migrate to a position in the separation column where the surrounding pH corresponds to its isoelectric point. There, in zwitterion form with no net charge, molecules of that substance cease to move in the electric field. Different amphoteric substances are thereby focused into narrow stationary bands.
Capillary isoelectric focusing (CIEF) is becoming recognized as a powerful separation technique for proteins and peptides due to its fast separation speed and ease of use for quantitative determination..sup.1 Until now most applications of CIEF have been done using commercial capillary electrophoresis (CE) instruments. These instruments have a 20-60 cm long capillary and an on-column UV absorption detector. When using these instruments for CIEF, all protein zones separated by the focusing process must be moved through the detection point of the on-column detector located at one end of the capillary. Isoelectric focusing can be done in commercial CE instruments by a "two step" method or a "one step" method.
In the "two step" method, the capillary inner wall is coated with non-cross-linked acrylamide to eliminate electroosmotic flow..sup.2 The capillary is first filled with mixture of sample and carrier ampholytes, and two electrolyte reservoirs connected to the capillary ends are filled with anolyte and catholyte, respectively. Then, a DC voltage is applied across the capillary. After the focusing process finishes, all protein zones are stationary inside the capillary. Finally, a mobilization process is used to move all these zones through the detection point. Mobilization can be achieved by using hydrodynamic force.sup.2 or adding salts.sup.2,3 to one end of the capillary column.
In the "one step" method, an uncoated capillary is used as the separation column. A plug of sample and carrier-ampholytes solution is introduced into the capillary between anolyte and catholyte. Additives, such as methylcellulose, are mixed into sample and electrolyte solutions to increase viscosity of the solution and reduce the electroosmotic flow..sup.4,5 Upon application of a DC voltage, protein components are focused inside the capillary while the whole volume of solutions inside the capillary is mobilized slowly from the anodic end to the cathodic end.
The "one step" method appears to have advantages over the "two step" method since it overcomes many problems of the "two step" method, such as blind section in the capillary,.sup.3 and the instability of the coating on the capillary inner wall..sup.4 The "one step" method is more compatible with current commercial CE instruments.
However, CIEF performed in commercial CE instruments using mobilization process has problems. Mobilization causes uneven resolution along the capillary column, and makes the pH gradient non-linear so that isoelectric point (pI) values of protein samples are difficult to estimate from their elution times. The mobilization process lengthens analysis time, since focusing takes 2-6 minutes while mobilization usually takes 10-30 minutes..sup.3-5 During the whole process all protein zones remain focused at the positions where their pI values are the same as pH values..sup.3-5 Keeping proteins at high concentration and in a state of zero net charge for a long time increases their chance of precipitation. In the "one step" CIEF method, concentrations of carrier ampholytes and additives, and capillary length and sample plug length have to be optimized to adjust mobilization speed in order to obtain the best resolution for different samples..sup.4,5
We earlier found that the aforementioned problems arising from the mobilization process can be overcome directly through the use of a new on-line, real-time imaging detector to replace the conventional on-column detectors. Such new detector systems, developed by the present inventors, are the subject of a number of recent publications, which disclose: a universal refractive index gradient imaging detector,.sup.6,7 an optical absorption imaging detector,.sup.7,8 and a fluorescence imaging detector..sup.9 To accommodate the imaging detector system in the CIEF instruments of this new variety, instrument, short capillaries, 4 cm long were employed. Isoelectric focusing performed in 4 cm long capillaries showed the same resolution as for CIEF performed in 20-60 cm long capillaries..sup.7 Analyzing a protein sample only takes 2-4 minutes using the imaging detection system. Isoelectric points of components can be determined directly from their positions along the capillary column. The CIEF-imaging detector system was also developed into a multichannel separation and detection method,.sup.10,11 which overcomes the low sample throughput problem of conventional CIEF.
The sample injection procedure used with CIEF-imaging detector instruments is generally as follows. The mixture of sample and carrier ampholytes is first forced into the capillary with a syringe, and then both electrolyte reservoirs connected to the two ends of the capillary are filled with anolyte and catholyte, respectively. Some skill and care are required to ensure that no bubbles are inside the capillary, and that the sample solution inside the capillary is not mixed with the electrolytes in the reservoirs..sup.7 Each time a sample is changed, the electrolyte in each of the two reservoirs must be changed. This sampling procedure takes about 2-5 minutes for single capillary instruments.